PROJECT SUMMARY A central mechanism of tumor drug resistance is the maintenance of tight junctions between malignant cells preventing penetration of molecules into the tumor microenvironment. We have generated junction openers (?JO?) that are small proteins that bind to desmoglein 2 (DSG2), a junction protein that is overexpressed in many cancers. Intravenous injection of JO increases tumor penetration and efficacy of many types of cancer therapy. Our studies have shown that the effective doses of chemotherapy can be reduced when the drugs are combined with JO. JO accumulates in tumor tissue as much as 100-fold above normal tissues making it a targeting mechanism to tumors. We have also published that application of JO has not been associated with toxicities in hDSG2 transgenic mice and that the co-administration of JO and chemotherapy was well tolerated in non-human primates. In phase 1 we were able to show that: (1) we can make JOC-x conjugates for a number of cancer treatment and imaging applications; and (2) that the JOC-x constructs retain their activity in tumor models. We now are motivated to build on the promising data generated in phase 1 and move these conjugates towards clinical testing by: (1) Preparing JOC-x for cGMP compliant production by process development, scaling, and writing manufacturing batch records; (2) Producing JOC-x conjugates with distinct functionalities to demonstrate utility and flexibility of the platform; and (3) Testing these JOC-x conjugates in animal models At the conclusion of the research proposed here we will have produced a conjugatable tumor tight junction opening candidate that can be used in a number of embodiments. Each of the products could become stand-alone therapeutics and be developed towards clinical testing.